Transfection of monoblastoid U937 cells with inducible nitric oxide synthase (NOS2) resulted in a human cell line that spontaneously produced large amounts NO, a mediator of vascular dysfunction in septic shock. After differentiation with phorbol-12-acetate-13-myristate (PMA), iNOS expressing cells produced increased amounts of TNF by a mechanism that was dependent on NO. This demonstrated the ability of endogenously generated NO to augment the inflammatory response (Blood, 1997). Likewise, overexpression of endothelial NOS (eNOS) was shown to upregulate TNFalpha production, but neither N-methyl-L-arginine, a NOS inhibitor, nor mutation of its L-arginine binding site (rendering it incapable of producing nitric oxide) blocked this effect (J Biol Chem, 2000). However, co-transfection with superoxide dismutase or deletion of the NADPH binding site of eNOS completely prevented eNOS from upregulating TNF production. Collectively, our results suggested that eNOS regulates inflammatory responses through both NO (J Immunol, 1994; J Biol Chem, 1997) and reactive oxygen species-based signal transduction pathways (J Biol Chem, 2000). Superoxide produced by eNOS was subsequently shown to upregulate TNFalpha via p42/44 MAPK activation (J Biol Chem, 2001). Sp1 was identified as a bidirectional NO sensor, down regulating eNOS in endothelial cells and upregulating TNF, both via proximal Sp promoter-binding sites (J Biol Chem, 2003). Direction (up or down) of the response was controlled by promoter sequences adjacent to the Sp1 binding site. Sickle cell disease was characterized by oxidant and inflammatory stress in the vasculature, even in the absence of an acute crisis (Blood, 2004). Circulatory stress in sickle cell disease was associated with gene expression and arginine metabolism abnormalities in platelets (Circulation, 2007). Anti-proliferative effects of NO that prevent the development of a chronic vascular injury phenotype was linked to p38 MAPK activation and p21 mRNA stabilization with subsequent down regulation of polo-like kinase 1 through a CDE/CHR proximal promoter site (BMC Genomics, 2005; J Biol Chem, 2006). Both NO and peroxisome proliferator-activated receptors (PPARs) protect the endothelium and regulate its function. Therefore, we tested for crosstalk between these signaling pathways using human umbilical vein and hybrid EA.hy926 endothelial cells (FASEB J, 2007). PPARgamma was activated by NO through a p38 MAPK dependent signal transduction pathway. This crosstalk mechanism may contribute to the anti-inflammatory and cytoprotective effects of NO in the vasculature and suggests new strategies for preventing and treating vascular dysfunction. The optimal activation of PPARgamma by thiazolidinediones (TZDs), drugs used in the treatment of type 2 diabetes mellitus, was found to similarly require p38 MAPK activation. TZDs, like homeostatic NO production, reduce markers of cardiovascular inflammation. We have subsequently identified a p38MAPK/acetyltransferase signal transduction mechanism that enhances the transcription of PPARgamma target genes (in preparation, 2012). PPARgamma agonist (rosiglitazone; RGZ) activation of p38 MAPK with downstream enhancement of PPARgamma signaling was linked to G-protein coupled receptor 40 (GPCR40). This finding defines PPARgamma signaling for the first time as a two receptor system. Cognate GPCR and nuclear receptor signaling networks may explain differences in safety and efficacy among nuclear receptor ligands and has implications for future drug development. Collectively, these findings suggest that PPARgamma may be a useful target for reducing endothelial dysfunction and vascular inflammation in septic shock and possibly chronic diseases such as PAH. Interrogation of our microarray database indicates that mineralocorticoid receptor (MR) is relatively overexpressed in endothelial cells and T-lymphocytes compared to monocytes and B-lymphocytes. Likewise, three other nuclear receptor family members, androgen receptor (AR), COUP-TFI and COUP-TFII were also found to be over-expressed in endothelial cells relative to leukocytes. In contrast, glucocorticoid (GR), a nuclear receptor target that can reverse shock but may lack overall benefit due to adverse effects on immune function, is expressed more uniformly across these cell types. Therefore, MR, AR, COUP-TFI and COUP-TFII may be useful targets for modulating endothelium inflammation. Using genome-wide expression profiling, COUP-TFII knockdown in a human endothelial cell line was shown to modulate 36% of all TNF-responsive genes (American Thoracic Society, abstract 2011). COUP-TFII suppression of TNFlapha-induced gene expression was associated with interferon signaling pathways (IRFs and STATs), while COUP-TFII amplification of TNFalpha responses was linked to NF-kappaB. Genome-wide gene expression differences in the peripheral blood mononuclear cells (PBMCs) of patients with PAH compared to healthy gender, age and ethnicity matched volunteers categorically identified alterations in inflammation, cell adhesion, cell motility, the cytoskeleton and apoptosis (American Thoracic Society, abstract 2011). Specific genes and canonical pathways overlapped with several previously proposed mechanisms and suggested novel therapeutic targets such as Ras, RhoA, integrin, focal adhesion kinase-1 (FAK), and p21-activated kinase (PAK). Recent work has investigated BMPR2 silencing in primary human pulmonary artery endothelial cells (PAECs) as an in vitro model of hereditary PAH. BMPR2 knockdown produces a phenotype with altered estrogen receptor expression, exaggerated inflammatory responses and dysregulated stress kinase signaling (Keystone Symposium on Pulmonary Arterial Hypertension 2012). In human endothelial cells both MR agonists and antagonists have been shown to repress NF-kappaB mediated gene transcription (Keystone Symposia on Nuclear Receptors, abstract, 2010; American Thoracic Society, abstract 2010), a finding with implications for the short-term use of mineralocorticoids in septic shock and long-term, early use of spironolactone in PAH. Based on this work, a pilot protocol to study early spironolactone therapy in PAH has been written and is undergoing scientific review.